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β-thalassemia and sickle cell disease are major human genetic health problem in many parts of the world. Available treatments are not satisfactory as none of them exhibit the optimal combination of safety, efficiency and convenience of use that would make them applicable to most hemoglobinopathy patients, especially to those who lack access to modern medical facilities. The observation that induction of γ-globin gene expression ameliorates the disease phenotype led to the proposal to induce γ-globin gene expression for the treatment of these diseases. Screening of a small molecule libraries (186000 compounds) has been done but new reagents with a higher HbF inducing effect and reduced cytotoxicity than those already known (butyrates, HU) were not found. Despite all the attempts made during the last 30 years, it is still not known how the γ-globin genes are switched off after birth, e.g. whether it is absence of activating factors or presence of suppressing factors or combination of both that leads to the down regulation of the γ-globin genes in the adult. There is only very limited information about the factors that are bound to the γ-globin promoters in vivo, certainly in its repressed state. It is likely that factors other than those described in the literature (at the start of this work) are essential for the suppression process. We therefore wanted to develop new strategies that specifically target fetal gene activation without cytotoxicity, widespread epigenetic alterations or difficult to manage side effects through the identification of the relevant transcription factors acting at the promoter. To this end we designed a strategy to isolate and identify protein factors bound in vivo to the suppressed human γ-globin gene promoter and to study their effect on γ-globin regulation (chapter 3). This targeted, in vivo single gene promoter chromatin purification has been carried out for the first time and is a completely novel strategy. To optimize such a purification protocol, different parameters have been tested including the use of various purification tags, reagents, buffers, etcetera (presented in chapter2). An alternative approach would be to identify target molecules within pathways that are involved in γ-globin gene expression. These pathways can be studied in the context of high and low HbF expressing β-thalassemia and sickle cell disease patients (chapter 4). There are many reports describing the induction of different HbF levels in response to hydroxyurea treatments in patients. We therefore wanted to understand the mechanism by which HU induces γ-globin in patients using expression analysis between so-called ‘responder’ and ‘nonresponder’ patients, i.e. between those showing high γ-globin production versus low γ-globin production. The results of this study are presented in chapter4. Finally in chapter 5 we present a novel set of genetic markers that is associated with increased γ-gobin expression in β-thalassemia patients followed by a discussion of future prospects in Chapter 6.
The research has been supported by NIH grant “Molecular basis of γ-globin gene activation”,
HL 073455, NGI, NOW, Landsteiner foundation for Blood Transfusion Research (NL), the
Center for Biomedical Genetics (NGI, NL) and the FP7 EuTRACC consortium of the European
The studies described in this thesis were partly supported by a grant from the BSIK
Innovation programme; Stem Cells in Development and Disease (SCDD, BSIK).
Financial support by SCDD for the publication of this thesis is gratefully acknowledged.
Financial support by the Erasmus MC, SCDD and J.E. Jurriaanse Stichting for the publication of
this thesis is gratefully acknowledged.
- γ- globin expression